The development of technologies allowing proteins to be synthesized at will are expected to contribute greatly, not only to the fields of the life sciences and biotechnology, but also to the design of nano-machines and the development of molecular components in such engineering fields as neural computing. Currently, genetic engineering techniques for introducing cloned DNA into living cells are widely used for protein synthesis, but exogenous proteins that can be produced by these methods are limited to molecules that are able to survive the life support mechanisms of their host. Meanwhile, advances in organic synthesis technology have made automatic synthesizers common, but while peptides comprising a few dozen amino acids are routinely synthesized, chemical synthesis of higher molecular weight proteins is currently extremely difficult, due to limitations in terms of the yield, side reactions, and the like. Furthermore, there has been strong ethical criticism in Europe and the United States of conventional using living organisms to produce proteins, or to search for novel molecules, and there is a concern that international regulations will become even stricter.
Cell-free protein synthesis is an example of a novel protein synthesis method capable of overcoming these problems, which applies biochemical procedures and attempts to make maximal use of the outstanding characteristics of living organisms. These methods provide biological systems for the translation of genetic information within artificial containers and, using nucleic acids which have been designed and synthesized as templates, reconstruct systems capable of incorporating the desired amino acids, including those which do not exist in nature. As these systems are not subject to the limitations of living organisms, it can be expected that an almost limitless range of protein molecules can be synthesized.
With regard to cell-free protein synthesis systems, it was reported 40 years ago that pulped cell sap retained the ability to synthesize protein, and various methods of doing this have been developed in the past. Currently, cell extracts derived from E. coli, wheat embryo and rabbit reticulocytes are widely employed in protein synthesis and the like.
The inventors have already shown, based on the findings of past research into ribosome inactivating toxins, that the extreme drop in protein synthesis activity seen in cell-free protein synthesis systems using wheat embryo extract were the result of a switch for an auto ribosome inactivation mechanism (cell suicide mechanism), which is programmed into the original cell as a defense mechanism against pathogenic microorganisms, and which is triggered by grinding the embryo (Madin, K. et al., Proc. Nat'l. Acad. Sci. USA, 97, 559-564 (2000)). Then, it was demonstrated that protein synthesis reactions using wheat embryo extract that was prepared by a novel method, wherein tritin activity and the like were eliminated from embryo tissues, exhibited good protein synthesis characteristics over a long period of time (Madin, K. et al., Proc. Nat'l. Acad. Sci. USA, 97, 559-564 (2000), JP-2000-236896-A).
However, in general, in the preparation of cell extracts for cell-free protein synthesis and performing translation reactions in the cell-free protein synthesis systems, as highly reducing conditions are required, proteins having at least one intramolecular disulfide bond cannot be formed. Consequently, there was a problem in that proteins having at least one intramolecular disulfide bond, which were prepared by conventional cell-free protein synthesis systems, often did not take on a three-dimensional structure and did not, therefore, have the original functions of the protein.